1. Field of the Invention
The present invention relates to a two-photon probe for real-time monitoring of intracellular free zinc ions, a method for preparing the two-photon probe, and a method for real-time monitoring of intracellular free zinc ions using the two-photon probe. More specifically, the two-photon probe has the ability to detect zinc ions deep inside live cells for a long period of time, thus being suitable for real-time imaging of intracellular free zinc ions.
2. Description of the Related Art
Zinc is a vital component of enzymes and proteins. In the brain, a few millimoles of intracellular free Zn2+ is stored in the presynaptic vesicles, released with synaptic activation, and seems to modulate excitatory neurotransmission. To understand the biological roles of zinc, a variety of fluorescent probes derived from quinoline (TSQ, Zinquin, and TFLZn) and fluoroscein (FluZn-3, Znpyr, ZnAF, etc.) have been developed.
However, most of such fluorescent probes require rather short excitation wavelength or suffer from pH sensitivity. To visualize the biological activity deep inside the live tissue (>80 μm) without the interference of surface preparation artifacts, two-photon microscopy (TPM) utilizing two-photons with low excitation energy is very effective. In particular, TPM employing two near-infrared photons for the excitation offers a number of advantages, including increased penetration depth, localized excitation and prolonged observation time, over one-photon microscopy (OPM). However, two-photon probes having the above advantages and being capable of effectively monitoring intracellular zinc ions appear to be rare. Although a few pH-resistant sensors for Zn2+ have been reported, they require either microinjection for cellular applications or use significant amount of ethanol as the co-solvent due to the poor water solubility.
There has been no efficient two-photon probe for Zn2+ that satisfies all requirements, including sufficient water solubility to stain cells, high selectivity for Zn2+, significant two-photon cross section, pH-resistance, and high photostability, and can monitor intracellular free zinc ions.